Seven different iPSCs, two different clones of two healthy controls (CONTROL 1 and CONTROL 2), two clones of THDA (THDA1#5 and THDA1#17), two clones of THDB (THDB1#1 and THDB1#15), and one isogenic (isoTHDA1#17), were differentiated into dopaminergic neurons using a 30‐day protocol based on DAn patterning factors and co‐culture with mouse PA6 feeding cells to provide trophic factor support, with minor modifications (Sánchez‐Danés et al, 2012 (link)). Specifically, iPSCs were cultured in mTeSR commercial medium until they reached 80% confluence and then mechanically aggregated to form embryoid bodies (EBs), without using lentiviral vectors to express LMX1A transcriptional factor. EBs were cultured for 10 days in suspension in N2B27 medium, consisting of DMEM/F12 medium (GIBCO), neurobasal medium (GIBCO), 0.5× B27 supplement (GIBCO), 0.5× N2 supplement (GIBCO), 2 mM ultraglutamine (Lonza) and penicillin–streptomycin (Lonza). In this step, N2B27 was supplemented with SHH (100 ng/ml, Peprotech), FGF‐8 (100 ng/ml, Peprotech), and bFGF (10 ng/ml; Peprotech). Neural progenitor cells (NPCs) were then seeded on top of PA6 for 21 days in N2B27 medium, as described (Sánchez‐Danés et al, 2012 (link)). Studied cultures were fixed with PFA 4% and characterized for dopaminergic specificity and for cell morphology.
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